Bottom Line:
Importantly, this production of IL-12 occurs very rapidly and is independent of interferon gamma priming or of signals from T cells, such as CD40 ligand.IL-12 production by splenic DC is accompanied by an increase in number of DCs, as well as a redistribution to the T cell areas and the acquisition of markers characteristic of interdigitating dendritic cells.This model avoids the need to invoke a three-cell interaction for Th1 differentiation and points to the DC as both a sentinel for innate recognition and the dictator of class selection in the subsequent adaptive response.

ABSTRACTThe early induction of interleukin (IL)-12 is a critical event in determining the development of both innate resistance and adaptive immunity to many intracellular pathogens. Previous in vitro studies have suggested that the macrophage (MPhi) is a major source of the initial IL-12 produced upon microbial stimulation and that this response promotes the differentiation of protective T helper cell 1 (Th1) CD4+ lymphocytes from precursors that are primed on antigen-bearing dendritic cells (DC). Here, we demonstrate by immunolocalization experiments and flow cytometric analysis that, contrary to expectation, DC and not MPhi are the initial cells to synthesize IL-12 in the spleens of mice exposed in vivo to an extract of Toxoplasma gondii or to lipopolysaccharide, two well characterized microbial stimulants of the cytokine. Importantly, this production of IL-12 occurs very rapidly and is independent of interferon gamma priming or of signals from T cells, such as CD40 ligand. IL-12 production by splenic DC is accompanied by an increase in number of DCs, as well as a redistribution to the T cell areas and the acquisition of markers characteristic of interdigitating dendritic cells. The capacity of splenic DC but not MPhi to synthesize de novo high levels of IL-12 within hours of exposure to microbial products in vivo, as well as the ability of the same stimuli to induce migration of DC to the T cell areas, argues that DC function simultaneously as both antigen-presenting cells and IL-12 producing accessory cells in the initiation of cell-mediated immunity to intracellular pathogens. This model avoids the need to invoke a three-cell interaction for Th1 differentiation and points to the DC as both a sentinel for innate recognition and the dictator of class selection in the subsequent adaptive response.

Figure 5: IL-12 production by LOD in response to systemic administration of STAg is restricted to CD8α+N418+ DC. LOD prepared from groups of STAg- or PBS-injected C57BL/6 mice were triple stained for IL-12 p40, N418, and CD8α or for DEC-205, N418, and CD8α. (A and B). IL-12+ cells are only seen in STAg-injected animals and are all bright for N418 (box). (C and D) Gating on N418+ cells demonstrates that IL-12+ cells seen in response to STAg (box) are part of the CD8α+ DC subset. All CD8α+ N418+ cells were also positive for NLDC-145, as reported (16, 17). Data are representative of three independent experiments. Other data from the same experiment are shown in Table 1.

Mentions:
To demonstrate that the IL-12 p40+ cells were indeed DC, sections were double stained with anti–IL-12 p40 and N418, a marker for mouse DC (20). As shown in Fig. 4 G, IL-12+ cells were also positive for N418 (white arrow) although some N418+ cells did not appear to stain for IL-12, particularly those N418+ cells at the edge or outside the T cell area (Fig. 4 G, black arrow). Furthermore, staining of serial sections with different antibodies demonstrated that IL-12+ cells colocalized with cells positive for the NLDC-145 marker (18), also known as DEC-205 (24), which is highly expressed by IDC in situ (data not shown). To confirm these results and to allow more accurate immunophenotyping of IL-12–producing cells, LOD suspensions were prepared in Ca2+-free media from mice injected with STAg or from controls injected with PBS, and then analyzed by flow cytometry. A distinct subpopulation of LOD from STAg-injected but not from PBS-injected mice could be stained intracellularly with anti–IL-12 p40 antibodies (Fig. 5, A and B). All IL-12+ cells were also N418bright, confirming the observations from immunohistochemistry that N418−/dull splenic MΦ, which, like DC, are enriched in LOD, do not produce significant levels of IL-12 after exposure to STAg in vivo (Fig. 5 B). Remarkably, most IL-12–producing N418+ cells were also positive for CD8α (Fig. 5, C and D), a marker that, like DEC-205, is expressed by IDC (16, 17). 67% of all the CD8α+ N418+ DC in STAg-injected animals stained for IL-12; in contrast, only a few (12%) of the CD8α− N418+ DC were positive for IL-12 and the intensity of IL-12 staining of these cells was lower than that of the CD8+ DC (mean fluorescence 223 versus mean fluorescence 644; see Fig. 5 D). We conclude that high levels of IL-12 production in mouse spleen can be detected shortly after systemic administration of two different microbial products, STAg and LPS. This IL-12 production almost exclusively involves a large proportion of CD8α+ DEC-205+ IDC, under conditions in which production of the same cytokine by splenic MΦ cannot be detected.

Figure 5: IL-12 production by LOD in response to systemic administration of STAg is restricted to CD8α+N418+ DC. LOD prepared from groups of STAg- or PBS-injected C57BL/6 mice were triple stained for IL-12 p40, N418, and CD8α or for DEC-205, N418, and CD8α. (A and B). IL-12+ cells are only seen in STAg-injected animals and are all bright for N418 (box). (C and D) Gating on N418+ cells demonstrates that IL-12+ cells seen in response to STAg (box) are part of the CD8α+ DC subset. All CD8α+ N418+ cells were also positive for NLDC-145, as reported (16, 17). Data are representative of three independent experiments. Other data from the same experiment are shown in Table 1.

Mentions:
To demonstrate that the IL-12 p40+ cells were indeed DC, sections were double stained with anti–IL-12 p40 and N418, a marker for mouse DC (20). As shown in Fig. 4 G, IL-12+ cells were also positive for N418 (white arrow) although some N418+ cells did not appear to stain for IL-12, particularly those N418+ cells at the edge or outside the T cell area (Fig. 4 G, black arrow). Furthermore, staining of serial sections with different antibodies demonstrated that IL-12+ cells colocalized with cells positive for the NLDC-145 marker (18), also known as DEC-205 (24), which is highly expressed by IDC in situ (data not shown). To confirm these results and to allow more accurate immunophenotyping of IL-12–producing cells, LOD suspensions were prepared in Ca2+-free media from mice injected with STAg or from controls injected with PBS, and then analyzed by flow cytometry. A distinct subpopulation of LOD from STAg-injected but not from PBS-injected mice could be stained intracellularly with anti–IL-12 p40 antibodies (Fig. 5, A and B). All IL-12+ cells were also N418bright, confirming the observations from immunohistochemistry that N418−/dull splenic MΦ, which, like DC, are enriched in LOD, do not produce significant levels of IL-12 after exposure to STAg in vivo (Fig. 5 B). Remarkably, most IL-12–producing N418+ cells were also positive for CD8α (Fig. 5, C and D), a marker that, like DEC-205, is expressed by IDC (16, 17). 67% of all the CD8α+ N418+ DC in STAg-injected animals stained for IL-12; in contrast, only a few (12%) of the CD8α− N418+ DC were positive for IL-12 and the intensity of IL-12 staining of these cells was lower than that of the CD8+ DC (mean fluorescence 223 versus mean fluorescence 644; see Fig. 5 D). We conclude that high levels of IL-12 production in mouse spleen can be detected shortly after systemic administration of two different microbial products, STAg and LPS. This IL-12 production almost exclusively involves a large proportion of CD8α+ DEC-205+ IDC, under conditions in which production of the same cytokine by splenic MΦ cannot be detected.

Bottom Line:
Importantly, this production of IL-12 occurs very rapidly and is independent of interferon gamma priming or of signals from T cells, such as CD40 ligand.IL-12 production by splenic DC is accompanied by an increase in number of DCs, as well as a redistribution to the T cell areas and the acquisition of markers characteristic of interdigitating dendritic cells.This model avoids the need to invoke a three-cell interaction for Th1 differentiation and points to the DC as both a sentinel for innate recognition and the dictator of class selection in the subsequent adaptive response.

ABSTRACTThe early induction of interleukin (IL)-12 is a critical event in determining the development of both innate resistance and adaptive immunity to many intracellular pathogens. Previous in vitro studies have suggested that the macrophage (MPhi) is a major source of the initial IL-12 produced upon microbial stimulation and that this response promotes the differentiation of protective T helper cell 1 (Th1) CD4+ lymphocytes from precursors that are primed on antigen-bearing dendritic cells (DC). Here, we demonstrate by immunolocalization experiments and flow cytometric analysis that, contrary to expectation, DC and not MPhi are the initial cells to synthesize IL-12 in the spleens of mice exposed in vivo to an extract of Toxoplasma gondii or to lipopolysaccharide, two well characterized microbial stimulants of the cytokine. Importantly, this production of IL-12 occurs very rapidly and is independent of interferon gamma priming or of signals from T cells, such as CD40 ligand. IL-12 production by splenic DC is accompanied by an increase in number of DCs, as well as a redistribution to the T cell areas and the acquisition of markers characteristic of interdigitating dendritic cells. The capacity of splenic DC but not MPhi to synthesize de novo high levels of IL-12 within hours of exposure to microbial products in vivo, as well as the ability of the same stimuli to induce migration of DC to the T cell areas, argues that DC function simultaneously as both antigen-presenting cells and IL-12 producing accessory cells in the initiation of cell-mediated immunity to intracellular pathogens. This model avoids the need to invoke a three-cell interaction for Th1 differentiation and points to the DC as both a sentinel for innate recognition and the dictator of class selection in the subsequent adaptive response.